Interstrand DNA crosslinks (ICLs) linking the two strands of DNA are highly toxic because they are such efficient blocks to DNA replication and transcription. Such crosslinks are formed by clinically relevant chemotherapeutic agents, and from environmental sources. Human cells can repair some of these challenging lesions, but the detailed mechanisms of crosslink repair are unknown. The research proposed here makes use of systems to place a single ICL within DNA and analyze its repair and processing by human enzymes. Aim 1 is to make use of unique substrates and new assays in order to determine the involvement of specific nucleotide excision repair (NER) proteins and reactions in repair of a psoralen ICL. First, the involvement of NER enzymes in cleavage on either side of a crosslink will be tested by examining cell extracts defective in NER, with the prediction that NER-defective XP extracts will lack both incisions. XPC-HR23B appears to be an initial recognition factor for DNA distortions. We will test whether the purified XPC-HR23B complex can bind to DNA containing a single ICL, and compare this binding to other characterized distortions. In NER, an intermediate characterized step is formation of an open complex around a lesion, where DNA becomes transiently single-stranded. To test whether the DNA can open up on either side of a crosslink to form a preincision complex, chemical footprinting methods will be used. We will determine whether the 6 core NER factors XPA, XPC-HR23B etc. are sufficient for release of one arm of an ICL and if not, human cell extracts will be fractionated in order to purify additional factors. Aim 2 is to determine whether Y structures that model DNA replication forks or sites of transcription are substrates for ERCC1-XPF. We hypothesize that such Y-structures could form during DNA replication or transcription and form substrates for action by ERCC1-XPF to release one arm of a crosslink. To investigate this, model DNA replication forks containing double-stranded DNA on each arm of a Y structure will first be tested for suitability as a substrate for ERCC1-XPF. It is predicted that ERCC1-XPF will be able to cleave on both sides of the ICL, when appropriately positioned with respect to the fork. Model substrates with DNA or RNA polymerases stalled near the crosslink will also be tested. Aim 3 is to investigate the biochemical activities and cellular functions of POLQ family DNA polymerases with respect to several properties relevant to DNA repair. Purified POLQ and POLN, a new enzyme discovered in our laboratory, will be tested for the ability to bypass DNA damage, including an unhooked DNA crosslink. The fidelity of the enzymes will be measured. To investigate cellular functions of POLQ and POLN, siRNA inhibition of the enzymes will be carried out followed by tests for changed sensitivity to DNA crosslinking agents. Changes in cellular localization after DNA damage will be examined. POLN and POLQ will be isolated from transfected cells by immunoprecipitation, and co-purifying proteins will be analyzed to identify new factors involved in DNA crosslink repair. Such factors might prove to be useful targets in future efforts to inhibit repair of ICLs in tumor cells.